Comprehensive analysis of the transcriptional landscape of the human FMR1 gene reveals two new long noncoding RNAs differentially expressed in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome

The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5′-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called “Deep-RACE”, which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3′ region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00439-013-1356-6) contains supplementary material, which is available to authorized users

Functional Characterization of FMR4, a Trans- Acting Long Non-Coding RNA Associated with the Fragile X Locus

CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism (Fragile X Syndrome, FXS), or adult-onset neurodegeneration (Fragile X-associated Tremor/Ataxia Syndrome, FXTAS). However, the FMR1 locus is complex and encodes several long noncoding RNAs (lncRNAs), whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. As cis- regulation of FMR1 by FMR4 was not detected, we sought trans-regulated FMR4 targets in vitro. Gene expression and chromatin immunoprecipitation microarrays identified differentially expressed FMR4-responsive genes. Activities of the polyadenylated and chromatin-associated FMR4 in human neural precursor cells (hNPCs) included regulation of genes involved in cell cycling, differentiation, the ubiquitin-proteasome pathway and a G-protein coupled receptor subunit. FMR4 is expected to share a bidirectional promoter with FMR1, but expression of the two transcripts diverges during peak hNPC proliferation. FMR4 decreases, while FMR1 RNA increases, and these changes are accompanied by corresponding differential expression of FMR4 target genes. By regulating gene expression, FMR4 may promote cellular proliferation rather than differentiation, a role supported by S-phase marker assays. We also identified protein partners of FMR4 using MS2-tagged RNA-immunoprecipitation and mass spectrometry. We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions

Non-coding RNAs as direct and indirect modulators of epigenetic regulation

Epigenetic regulation of gene expression is an increasingly well-understood concept that explains much of the contribution of an organism's environment and experience to its biology. However, discussion persists as to which mechanisms can be classified as epigenetic. Ongoing research continues to uncover novel pathways, including the important role of non-protein coding RNA transcripts in epigenetic gene regulation. We know that the majority of human and other mammalian transcripts are not translated but that many of these are nonetheless functional. These non-coding RNAs (ncRNAs) can be short (<200 nt) or long (<200 nt) and are further classified by genomic origin and mechanism of action. We discuss examples of ncRNAs that interact with histone modifying complexes or DNA methyltransferases to regulate gene expression, others that are targets of these epigenetic mechanisms, and propose a model in which such transcripts feed back into an epigenetic regulatory network

RNAi Joins the “Singles Club”

Biotechnology &amp; Applied MicrobiologyGenetics &amp; HeredityMedicine, Research &amp; ExperimentalSCI(E)1EDITORIAL MATERIAL112010-20112

Changes in expression of the long non-coding RNA FMR4 associate with altered gene expression during differentiation of human neural precursor cells

CGG repeat expansions in the Fragile X mental retardation 1 ( FMR1 ) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism Fragile X Syndrome (FXS), or adult-onset neurodegeneration Fragile X-associated Tremor/Ataxia Syndrome. However, the FMR1 locus is complex and encodes several long non-coding RNAs, whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4 , which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4 , thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. Since the two transcripts do not exhibit cis -regulation of one another, we examined the potential for FMR4 to regulate target genes at distal genomic loci using gene expression microarrays. We identified FMR4 -responsive genes, including the methyl-CpG-binding domain protein 4 ( MBD4 ). Furthermore, we found that in differentiating human neural precursor cells, FMR4 expression is developmentally regulated in opposition to expression of both FMR1 (which is expected to share a bidirectional promoter with FMR4 ) and MBD4 . We therefore propose that FMR4 ’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions

The long non-coding RNA FMR4 promotes proliferation of human neural precursor cells and epigenetic regulation of gene expression in trans

Triplet repeat expansions in the Fragile X mental retardation 1 (FMR1) gene cause either intellectual disability and autism, or adult-onset neurodegeneration, with poorly understood variability in presentation. Previous studies have identified several long noncoding RNAs (lncRNAs) at the FMR1 locus, including FMR4. Similarly to FMR1, FMR4 is silenced by large-repeat expansions that result in enrichment of DNA and histone methylation within the shared promoter and repeat sequence, suggesting a possible role for this noncoding RNA in the pathophysiology of Fragile X. We therefore assessed the functional role of FMR4 to gain further insight into the molecular processes in Fragile X-associated disorders. Previous work showed that FMR4 does not exhibit cis-regulation of FMR1. Here, we found that FMR4 is a chromatin-associated transcript and, using genome-wide chromatin immunoprecipitation experiments, showed that FMR4 alters the chromatin state and the expression of several hundred genes in trans. Among the genes regulated by FMR4, we found enrichment for those involved in neural development and cellular proliferation. S-phase marker assays further demonstrated that FMR4 may promote cellular proliferation, rather than differentiation, of human neural precursor cells (hNPCs). By establishing this novel function for FMR4 in hNPCs, we lend support to existing evidence of the epigenetic involvement of lncRNA in nervous system development, and increase our understanding of the complex pathogenesis underlying neurological disorders associated with FMR1 repeat expansions

The Effect of Variation in Expression of the Candidate Dyslexia Susceptibility Gene Homolog Kiaa0319 on Neuronal Migration and Dendritic Morphology in the Rat

We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct (“rescue”), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non–transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non–cell autonomous mechanisms. Of the Kiaa0319 shRNA–transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites